Objectives: To ensure the microbiological safety of groundwater, it was confirmed whether waterborne pathogenic bacteria in groundwater samples tested positive for total coliforms in the Chungcheongnam-do Province region. Methods: Total colony counts, total coliforms and fecal coliforms were tested according to the process mandated by the drinking water quality testing standards of Korea. DNA was extracted from the samples, tested positive for total coliforms, and then subjected to real-time PCR to detect waterborne pathogenic bacteria. Results: A total of 115 samples were inadequate for drinking water. Thirty-one cases (27%) showed positive for fecal coliforms and nine cases (7.8%) showed total colony counts exceeding drinking water standards. Twenty-seven cases (23.5%) showed three items (total colony counts, total coliforms and fecal coliforms). Using the real-time PCR method, waterborne pathogens were detected in 57 cases (49.6%) in 115 samples. Seventy-eight cases of waterborne pathogenic bacteria were detected (including duplications): 27 cases of pathogenic E. coli (EPEC (19), ETEC (5), EHEC (1), EAEC (1) and EIEC (1)); 45 of Bacillus cereus; two of Yersinia spp.; two of Salmonella spp.; one of Staphylococcus aureus; one of Clostridium perfringens. Conclusion: The real-time PCR method can offer rapid and accurate detection of waterborne pathogenic bacteria. Therefore, this assay could be an alternative to conventional culture methods and can further ensure the microbiological safety of groundwater.